Abstract
The MultiSite Gateway cloning system, based on site-specific recombination, enables the assembly of multiple DNA fragments in predefined order, orientation, and frame register. To streamline the construction of recombinant genes for functional analysis in plants, we have built a collection of 36 reference Gateway entry clones carrying promoters, terminators, and reporter genes, as well as elements of the LhG4/LhGR two-component system. This collection obeys simple engineering rules. The genetic elements (parts) are designed in a standard format. They are interchangeable, fully documented, and can be combined at will according to the desired output. We also took advantage of the MultiSite Gateway recombination sites to create vectors in which two or three genes can be cloned simultaneously in separate expression cassettes. To illustrate the flexibility of these core resources for the construction of a wide variety of plant transformation vectors, we generated various transgenes encoding fluorescent proteins and tested their activity in plant cells. The structure and sequence of all described plasmids are accessible online at http://www.psb.ugent.be/gateway/. All accessions can be requested via the same Web site.
Highlights
The MultiSite Gateway cloning system, based on site-specific recombination, enables the assembly of multiple DNA fragments in predefined order, orientation, and frame register
The MultiSite Gateway technology has been developed for the simultaneous cloning of multiple DNA fragments in a versatile format (Cheo et al, 2004; Sasaki et al, 2004; Magnani et al, 2006), and plant binary T-DNA vectors have been devised for MultiSite Gateway protocols (Karimi et al, 2005)
From 5# to 3# relative to transcription, the promoters or enhancer motifs are flanked by the attL4 and attR1 recombination sites, open reading frames (ORFs) by the attL1 and attL2 or attR2 and attL3 sites, and terminators by the attR2 and attL3 sites
Summary
Several cloning systems are available for the transfer of segments between double-stranded DNA molecules that bypass the multistep protocols involving restriction enzyme and ligase reactions These systems are based on recombinases that recognize specific DNA sequences long enough not to occur by chance but short enough not to interfere with the function of the cloned elements. The MultiSite Gateway technology has been developed for the simultaneous cloning of multiple DNA fragments in a versatile format (Cheo et al, 2004; Sasaki et al, 2004; Magnani et al, 2006), and plant binary T-DNA vectors have been devised for MultiSite Gateway protocols (Karimi et al, 2005) In this context, two, three, or more fragments, flanked by compatible attL and/or attR sites in so-called entry clones, can be recombined in predefined order and orientation into a destination vector in a single LR clonase reaction.
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